Gene 543 (2014) 28–33

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Direct gene disruption by TALENs in medaka embryos☆ Tiansu Wang 1, Yunhan Hong ⁎ Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543, Singapore

a r t i c l e

i n f o

Article history: Received 18 December 2013 Received in revised form 11 March 2014 Accepted 4 April 2014 Available online 5 April 2014 Keywords: dnd Gene disruption Genome editing Gene targeting TALEN

a b s t r a c t Targeted gene disruption (GD) is powerful for generating genetic alterations in animal genomes. Engineered endonucleases such as zinc finger nucleases and transcription activator-like effector nucleases (TALENs) allow for GD directly in animal embryos to achieve germline transmission. Here we report procedures and parameters of TALEN-mediated GD in the fish medaka by using a germ cell-specific gene dnd as a model. Embryos at the 1-cell stage were microinjected with synthetic TALEN mRNAs and examined for the survival rate and GD efficiency. Medaka embryos can tolerate a high dosage of TALEN-mRNA injection and exhibit a steadily increasing GD efficiency with increasing mRNA dosages before peaking at 100 ng/μl. This dosage produced ~24% efficiency for somatic GD. Some of the animals from manipulated embryos developed into fertile female and male. Most importantly, four fish (3 males and 1 female) examined by progeny-test were able to produce GD-bearing male and female gametes for germline transmission to F1 generation at ~10% efficiency. Therefore, TALEN is proficient for somatic and germline GD in medaka embryos, and disruption of one dnd copy does not compromise somatic development and gamete production. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Gene targeting (GT) has long been the ‘gold standard’ for determining a certain gene's function in mammals. It utilizes homologous recombination (HR) machineries to introduce designed genomic modifications into cells. With GT, researchers can not only precisely target a specific locus but also have complete control of the way the locus is modulated (Capecchi, 2005). Because murine embryonic stem (ES) cells are germline-competitive, the application of GT on ES cells has enabled the creation of germline chimeras (Thomas and Capecchi, 1987). This technology has become a routine in mice since then, and produced thousands of transgenic mouse lines, which characterized specific gene functions and established human disease models (Capecchi, 2005). However, GT is limited to only mouse and rat, because the frequency of HR is inherently low and the method has to be performed in germ-line-competitive pluripotent cell lines (Deng and Capecchi, 1992). Engineered endonucleases can generate DNA double-strand breaks (DSBs) in a sequence-specific manner, stimulating DNA repairing through either non-homologous end joining (NHEJ) or HR. NHEJ introduces minor genetic alterations such as additions, deletions and mismatches (Jasin, 1996), whereas HR may lead to gene replacement Abbreviations: GD, gene disruption; Hm, homoduplex; Ht, heteroduplex; TALEN, transcription activator-like effector nuclease. ☆ Authors' contributions: Tiansu Wang did the research. Yunhan Hong designed the research and wrote the paper. ⁎ Corresponding author at: Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543, Singapore. E-mail address: [email protected] (Y. Hong). 1 Contains part of PhD thesis work.

http://dx.doi.org/10.1016/j.gene.2014.04.013 0378-1119/© 2014 Elsevier B.V. All rights reserved.

between a chromosomal gene and its introduced homolog (Bibikova et al., 2001). Engineered endonucleases used for GD include the zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR/Cas nuclease. These enzymes have been successfully used in zebrafish (Doyon et al., 2008; Xiao et al., 2013; Zu et al., 2013). The fish medaka (Oryzias latipes) is an excellent model for investigating fundamental questions in genetics and developmental biology (Shima and Mitani, 2004; Wittbrodt et al., 2002). It is well-suited to study stem cell biology and to develop genome editing technologies in lower vertebrates; We have recently reported GT in medaka ES cells (Guan et al., 2013; Yan et al., 2013) and direct gsdf GD in medaka embryos by using ZFNs (Chen et al., 2012; Zhang et al., 2013). In this organism, TALENs have been reported successful for GD on the dj-1 locus (Ansai et al., 2013). This study was aimed at the establishment of procedures and parameters for TALEN-mediated GD in medaka embryos by using germ cell-specific dnd as an independent model system. We show that TALENs can efficiently generate a high efficiency of GD in somatic and germ cells of the developing medaka embryo. 2. Materials and methods 2.1. Fish and chemicals Fish work was strictly adhered to the guidelines on Care and Use of Laboratory Animals of the National Advisory Committee for Laboratory Animal Research in Singapore and approved by this committee (permit number 67/12). Medaka strain Hd-rR was maintained under an artificial photoperiod of 14-h/10-h light/darkness at 26 °C as described (Hong

T. Wang, Y. Hong / Gene 543 (2014) 28–33

et al., 2011). Chemicals were from Sigma and enzymes from Promega and TaKaRa, unless otherwise indicated. 2.2. Plasmids A pair of TALEN expression vectors, pTN1dnd and pTN2dnd was designed to target a specific locus at the third exon of medaka dnd gene, and constructed with golden gate shuffling method (Cermak et al., 2011). The TALEN backbone contains a 290-aa N-terminus and a 246aa C-terminus, which is followed by endonuclease Fok I. Each of the plasmids contains a TALEN repeat variable di-residue (RVD) array and binds to one of the two recognition sequences, which are (pTN1dnd, TGGGACGGTCCCCCACC) and (pTN2dnd, TCTGGCTGATAAAGACC). The TALEN recognition sequences are separated by a spacer of 15 bp (TGGGGCGCGCTGTGA) for TALEN dimer formation. The recognition sequences within the target site amount up to 34 bp in length. 2.3. RNA synthesis and microinjection Synthetic mRNAs were generated by using ApaI-linearized TALEN expression vectors as templates and the mMessage mMachine T7 Ultra Kit (Ambion). Synthetic mRNAs were stored at −80 °C. 2.4. Microinjection Medaka embryos were injected at 1-cell stage. Briefly, the 1-cell embryos received 1 nl volume per injection, respectively. Capped TALEN mRNAs were injected in gradient concentrations: 25, 50, 100, 200, 400 ng/μl. The survival rates of embryos injected with different TALEN mRNA concentrations were monitored until hatching. DEPC-water was used as control injection.

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2.7. Cloning and sequencing PCR products before or after polyacrylamide gel enrichment were TA-cloned into pGEM-T Easy vectors (Promega). DNA mini-preps were prepared following the standard alkaline lysis protocol and used for sequencing on the 3130xl Sequencer (Applied Biosystems). Sequence analyses were performed by using the Vector NTI and DNAman software. 3. Results 3.1. Experimental design The germ cell-specific dnd was chosen for establishing procedures in this study, as its expression occurs exclusively in germ cells and its disruption would have little adverse effect on the survival and somatic development of embryos and animals (Liu et al., 2009). The medaka dnd consists of 5 exons, with exon 3 encoding the RNA recognition motif essential for the function. Two vectors, namely pTN1dnd and pTN2dnd, were constructed to express two TALENs targeting the medaka dnd gene (Fig. 1). They are shown for nucleotide sequence in Figs. S1 and S2, and protein sequence in Fig. S3. The TALENs were designed to recognize and target a 34-bp sequence within exon 3 (Fig. 1). This target sequence is present only once in the medaka genome by a blast search (http://www.ensembl.org/Oryzias_latipes/Info/Index), which predicts the specificity of TALENs for cleavage and GD. Upon introduction of the TALEN mRNAs, repairing upon DNA breaks will introduce subtle addition and/or deletion to the target site, usually producing frame shift mutations or truncated translation and thus leading to the generation of dnd GD alleles. Two primers were designed for PCR genotyping (Fig. 1, bottom), which flank a 196-bp fragment spanning the target site (Fig. S4A).

2.5. Genomic DNA extraction 3.2. Dose-dependent survival Genomic DNA was isolated from 7-dpf embryos and the adult caudal fin (Chen et al., 2012; Hong et al., 1998). Groups of 25 embryos were collected in 1.5-ml Eppendorf tubes and stored at −80 °C. Frozen embryos were homogenized by using a plastic pestle and treated with 200-μl TNES-6U buffer (10 mM Tris–HCl, pH 7.5; 125 mM NaCl; 10 mM EDTA; 1% SDS; 6 M Urea) and 5 μl of proteinase K solution (20 mg/ml) overnight at 37 °C. Following extraction with 300 μl of phenol/chloroform mixture, DNA was ethanol-precipitated and dissolved in 50 μl of TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) (Hong et al., 1998). 2.6. PCR and gel electrophoresis PCR was run in a 20-μl volume containing 25 ng of medaka genomic DNA as the template and using two primers (forward, TTTACATTTCGT GTTCAGTGTGG; reverse, GTGCCGTATTTGGCGTAAG) for 35 cycles (94 °C for 15 s, 60 °C for 15 s and 72 °C for 30 s). PCR products were separated on 10% polyacrylamide gels in 1× TBE buffer with the MiniProtean electrophoresis unit (Bio-Rad Laboratories) at 100 V for 2 h at room temperature. Gels were submerged in 1 × TBE buffer containing 0.05 μg/ml ethidium bromide for 15 min. Gels were documented on a bioimaging system (Vilber Lourmat). In this assay system, DNA of both WT and MT alleles will form – besides WT–WT and MT–MT homoduplexes (Hm) – WT–MT heteroduplex (Ht) that can easily be distinguished on PAGE due to considerably lagged migration. Enrichment of desired DNA bands includes gel recovery and successive PCR (grsPCR). DNA bands were cut from polyacrylamide gel under UV illuminator, submerged into 100 μl of TE buffer and smashed into pieces with 200-μl pipette-tips. After overnight incubation at 37 °C, the DNA-containing supernatant was subjected to successive PCR under the same conditions. The enriched PCR products were visualized on a polyacrylamide gel. Band intensity and abundance on polyacrylamide gel were quantified by using the Gel-Pro analyzer software.

Two key parameters of GD experimentation are the survival rate and GD efficiency, which have been shown to correlate with dosages of engineered nucleases such as ZFNs (Zhang et al., 2013). In order to establish a procedure for TALEN-mediated dnd GD, these parameters were firstly examined. The survival rates at the hatching stage were 93%, 82%, 83% and 71% in embryos injected with 25, 50, 100 and 200 ng/μl of TALEN mRNAs, respectively. 97% of water-injected control embryos survived observation. Notably, embryos injected with an extremely high dosage (400 ng/μl) exhibited abnormal development and massive death, and thus an extremely low survival rate of 0.5% (Table 1). 3.3. Dose-dependent GD efficiency in embryos The effects of different dosages on GD efficiency were tested. As expected, control embryos produced only an Hm band, whereas embryos injected with TALEN mRNAs generated not only an Hm band for the WT allele but also a varying number of additional Ht bands. The number and intensity of Ht bands increased with the dosage of injected TALEN mRNAs (Fig. 2A). This was more evident after enrichment for rare MT alleles by the gel recovery and subsequent PCR procedure (Fig. 2B). A densitometry-based semi-quantitative analysis by using the GelPro software was performed to examine the effect of TALEN mRNA dosage on the relative abundance of GD alleles. The relative intensities of Ht bands were found to be 10.6, 21.3 and 32.4% in embryos injected with 25, 50 and 100 ng/μl of TALEN mRNAs, respectively (Figs. 2C and S4C). This revealed a correlation between GD abundance and TALEN mRNA dosages. In parallel, the numbers of Ht variants were found to be 2, 3, and 8 in embryos injected with 25, 50 and 100 ng/μl of TALEN mRNAs, respectively (Figs. 2C and S4D), again revealing a correlation between the number of GD events and TALEN mRNA dosage. An exception to

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T. Wang, Y. Hong / Gene 543 (2014) 28–33

Fig. 1. TALEN expression vectors and target site. Shown is the map of pTN1dnd and pTN2dnd (top), the TALEN target site (middle; open triangle) and targeted alleles (bottom). The target site consists of two recognition sequences (red and blue) flanking a spacer. Each of TALENs has 16 RVDs (red and blue) and – together with a T at the 5′ start – recognizes a total of 17 bp. The target site contains two 17-bp recognition sequences and a 15-bp spacer and is located within exon 3 of gene dnd. TALENs generate a double-strand DNA break (asterisk) within the target site to initiate DNA repair and thus yield GD alleles with subtle additions and deletions within or near the target site. Arrowheads depict primers to amplify a 196-bp PCR product flanking the target site (for sequence information see Fig. S4A).

both observations was seen in embryos injected with an extremely high TALEN mRNA dosage (≥200 ng/μl), where a decrease rather than an increase in intensity and number of Ht bands was recorded (Fig. 2C). Embryos injected with 100 ng/μl of TALEN mRNAs were then individually sampled at 7 dpf for genotyping by PAGE. 11 out of 20 embryos examined produced obvious Ht bands (Fig. 3A), demonstrating the generation of GD alleles at 55% efficiency. Taken together, the TALEN approach shows a dose-dependent proficiency for direct GD in medaka

Table 1 Dose-dependent survival rate of TALEN-injected embryos.1 [TALEN mRNA] (ng/μl each)

0 25 50 100 200 400

Embryos injected, n

30 44 66 95 111 64

Survivor, n (%)2 Gastrula

Hatching

Fry

30 (100) 44 (100) 62 (94) 89 (94) 103 (93) 46 (72)

30 (100) 43 (98) 56 (85) 81 (85) 91 (82) 7 (11)

29 (97) 41 (93) 54 (82) 79 (83) 79 (71) 3 (0.5)

1 The relationship between survival rate and various doses of TALEN mRNA was shown. TALEN mRNAs were injected at 25, 50, 100, 200 and 400 ng/μl. Water injection served as a negative control. 2 Percentage values were derived by comparison to the number of embryos injected.

embryos, and the dosage of 100 ng/μl appears to be satisfactory in terms of survival rate and GD efficiency. 3.4. GD efficiency in adults A total of 400 medaka embryos at the 1-cell stage were injected with 100 ng/μl of TALEN mRNAs. Of these injected embryos, 122 developed into adult F0 fish. DNA was extracted from fin clips and subjected to genotyping by PCR and PAGE. It was found that 29 of the F0 fish exhibited Ht bands, indicating that they were putative founder animals (Fig. 3B). Various Ht bands formed between GD and WT alleles showed retarded mobility compared to the Hm bands. Besides GD-containing Ht Table 2 Germline transmission of GD alleles. Founder

#1 #3 #7 #19 #58 1 2

Mutant alleles1

Frequency

Fin

Germline

A4D7 N/D2 D5 D4/A14D8 A4D15

A3 D10 N/D A1D11 D8

A, addition; D, deletion. N/D, not detected.

10.7% (9/84) 7.7% (2/26) N/D (0/28) 11% (2/18) 13% (2/15)

T. Wang, Y. Hong / Gene 543 (2014) 28–33

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Fig. 2. Dose-dependent GD efficiency in medaka embryos. Embryos were injected at the 1-cell stage with indicated doses of TALEN mRNAs. Genomic DNA was isolated from 25 pooled fry and PCR products were analyzed on PAGE. (A) GD alleles in heteroduplex bands. (B) GD alleles in heteroduplex bands following enrichment by gel recovery and successive PCR (grsPCR). DNA size markers are shown to the right. Heteroduplex (Ht) bands migrate slower and are above homoduplex (Hm) bands. (C) Dose-dependent abundance of GD alleles.

Fig. 3. Genotyping of GD alleles. Embryos injected with 100 ng/μl mRNAs were analyzed for heteroduplex formation on PAGE. (A) Individual embryos at 7 dpf. (B) Representative F0 adults from injected embryos. ctl, non-injected control fish. (C) F1 progeny adults from founder fish female 1 and male 1. Heteroduplex (Ht) bands containing GD alleles, and homoduplex (Hm) WT allele (WT) and GD alleles (GD) are indicated. Arrowheads depict Hm of GD alleles in (B and C). DNA size markers are shown to the left.

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T. Wang, Y. Hong / Gene 543 (2014) 28–33

bands, a faint GD-Hm band may also be visible in certain F0 fish such as fish #14 and male 2. Taken together, injection with 100 ng/μl of TALEN mRNAs is capable of generating GD alleles at a high efficiency of 23.8% in adult fish. 3.5. Germline transmission F0 fish #1, #3 #19 and #58 (mentioned as female 1, male 1, male 2 and male 3 afterwards, respectively) derived from TALEN-injected embryos grew to adulthood. They were crossed with normal fish of opposite sexes, and the resultant progeny was examined for germline transmission. All of them were found to be founders, as they transmitted a GD allele to their F1 progeny. Specifically, female 1 was a female and transmitted a GD allele to 9 out of 84 progenies, thus demonstrating an ~11% germline transmission rate. Male 1 transmitted GD allele to 2 out of 28 F1 progenies, thus demonstrating an ~7% germline transmission rate. Male 2 has transmitted a GD allele to 2 out of 18 F1 progenies, producing an ~11% germline transmission rate. Male 3 transmitted a GD allele to 2 out of 15 F1 progenies, giving an ~13% rate of germline transmission (Table 2). These data suggest that TALEN is able to produce a high GD efficiency in both the soma and germline. Importantly, the relative abundance or intensity of GD PCR products (i.e. Ht band and Hm band of the GD allele) was comparable to that of the WT PCR product (Hm of WT allele) in the progeny of both female 1 and male 1 (Fig. 3C). This observation is in accordance with germline transmission of GD alleles and production of heterozygous F1 animals. Taken together, TALENs are capable of generating germline GD. 3.6. Diversity of GD alleles GD events were validated by sequencing of cloned PCR products from gel-recovered Ht bands. GD alleles with various alterations were identified, which fall into three major categories, namely alleles with added nt (addition), deleted nt (deletion), or a combination of addition, deletion plus substitution (compound alteration). Up to 9 different GD alleles were found in 6 F0 fish (Fig. 4). Fish #13, #15 and male 2 have deletion GD alleles missing 14, 11, and 4 nt, respectively. Fish male 2 and male 3 have compound GD alleles containing both addition and deletion. Notably, the progeny of female 1 produced two Ht bands besides a major Hm band of the WT allele (Fig. 3C), indicating the presence of one or two altered alleles, since one loop formed by mismatching PCR products may generate one or two Ht bands depending on DNA configuration (Chen et al., 2012). Sequencing of cloned PCR products revealed

the presence of one and same allele, namely A4D7, in all of the 9 progenies. In A4D7, a region of 7 nt within the target site is replaced by 4 nt, leading to a net loss of 3 nt (Fig. 4). Notably, this germlinetransmitted allele is different from the somatic GD allele A3 in the fin of the founder (Fig. 4), indicating that they were products of two independent GD events. The progeny of male 1 produced one Hm band and one Hm band of the GD allele besides an Hm band of the WT allele. Sequencing revealed that the GD allele in the progeny had a deletion of 10 nt (D10). It deserves to note that male 1 possesses only the WT allele but no detectable GD event in the fin (Fig. 4), indicating that allele D10 was generated after the separation of the germline from somatic lineages leading to the fin. 3.7. Fertility of dnd-disrupted gametes Fish derived from TALEN-injected embryos often developed into fertile female and male adults. GD transmission from female 1 and male 1 founder fish provoked a closer inspection on the sequence of altered dnd alleles. In female 1, the germline-transmitted allele A3 has 3 more nt, and would thus not affect the translation into a full length Dnd variant containing one more amino acid. In male 1, the germline-transmitted allele D10 has 10 fewer nt and thus represents a frame shift mutation affecting the translation. F1 progeny fish heterozygous for either maternally inherited A3 allele or paternally inherited D10 allele were found to have a normal growth to their control siblings. 4. Discussion In this study, we have established procedures and parameters for TALEN-mediated GD on the chromosomal loci dnd in medaka embryos via mRNA injection into medaka embryos. We show that the survival rate and GD efficiency are dependent on the dose of injected TALEN mRNAs. Specifically, TALENs at an optimal dose of 100 μg/μl is able to produce a high survival rate of up to 85% and a GD efficiency of 55% at the hatching stage. The GD-positive embryos develop normally into fertile female and male adults capable of transmitting GD alleles into 10% progeny of the F1 generation. Therefore, the TALEN approach is proficient for GD in the soma and germline of medaka embryos. These data provide valuable information for TALEN-mediated GD in medaka as well as other fish species. In medaka, direct GD by using the ZFN approach has been reported on transgene gfp (Ansai et al., 2012) and gsdf as a chromosomal gene (Zhang et al., 2013). In both studies, ZFNs produce a wide variety of GD alleles with subtle sequence alterations including additions,

Fig. 4. Sequence analysis of GD alleles. DNA was extracted from the fin of adult fish from TALEN-injected embryos or F1 progeny fish. Sequences shown here are for the WT and GD alleles within the target site with the spacer being highlighted. Small letter cases indicate introduced sequence substitutions. A, addition; D, deletion. Numerals after A or D indicate the numbers of nt added or deleted. Notably, male 2 had two different GD alleles in the fin, female 1 had a GD allele in the fin. All of female 1, male 2 and male 3 transmitted a different allele to the F1 progeny. Male 1 transmitted a GD allele, namely D10, to the F1 progeny without showing a detectable GD allele in the fin.

T. Wang, Y. Hong / Gene 543 (2014) 28–33

deletions and compound changes. In this study, TALENs targeting on the chromosomal locus dnd can also produce similar diversity of GD alleles. These results suggest that the TALEN and ZFN approach share a common ability to generate a wide variety of allelic alterations in medaka. In medaka, gsdf-targeting ZFNs under optimized conditions have produced a 58% GD efficiency and a 48% survival rate (Zhang et al., 2013). In this study, dnd-TALENs at an optimized dosage have given rise to a 55% GD efficiency and an 85% survival rate. More importantly, our results in this study demonstrate that all the four fish from TALEN-injected are capable of transmitting a GD allele to 7.7–13% of the F1 progeny, suggesting a high efficiency in generating germline transmitters. These twin studies appear to point to the advantage of TALENs over ZFNs in terms of GD efficiency, survival and ease with which that TALENs are engineered. This notion is supported by a recent report that the TALEN approach is highly efficient for dj-1 GD (Ansai et al., 2013). The GD efficiency observed in this study peaks at 32% and the germline transmission rate ranges from 7.7% to 13%. A nearly 100% GD efficiency and a germline transmission rate of 43.8%–100% have been obtained by using TALEN at 50–300 ng/μl on dj-1 (Ansai et al., 2013). Multiple factors may account for such differences in efficiency. For example, it is known that the efficiency of TALENs is depending on genomic context, with different target genes producing varying efficiencies (Cade et al., 2012). In addition, other differences in the structure of TALENs such as the lengths of N- and C-termini may also influence the efficiency. Collectively, TALENs are efficient for direct gene disruption in medaka embryos. The zebrafish dnd encodes an RNA binding protein cell-autonomously essential for primordial germ cell migration and survival (Weidinger et al., 2003). In medaka, dnd RNA expression is specific for embryonic and adult germ cells (Liu et al., 2009). Since germ cells are dispensable for embryonic and adult development, loss of Dnd functions would have little adverse effect on somatic development. In this study, the target site is within exon 3 that encodes a part of the RNA recognition motif essential for Dnd activity. TALEN-mediated GD within and around this target site is expected to produce loss-of-function alleles. It has been reported that dnd knockdown is able to produce sterility because of severe PGC migration defects (Weidinger et al., 2003). Germline transmitters in our system were highly mosaic in terms of a disrupted dnd allele, and almost all of the cells containing this allele are expectedly heterozygous before the completion of meiosis and thus capable of development during embryonic and post-hatching development. However, such a disrupted allele will stand alone in meiotic products. The fact that female, and more importantly, male founders are able to transmit a mutant dnd allele to F1 generation strongly suggests that genomic dnd is dispensable for germ cell development during the post-meiotic haploid phase. Future work will determine the role of Dnd on germ cell development in homozygous embryos and animals. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.04.013.

Conflict of interest The authors declare that there are no conflicts of interest.

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Acknowledgments We thank J. Deng for fish breeding and Xi Zhang for discussion. This work was supported by a grant from the National Research Foundation Singapore to Y.H. (NRF-CRP7-2010-03) and the scholarship from the National University of Singapore to T.W. References Ansai, S., Ochiai, H., Kanie, Y., Kamei, Y., Gou, Y., Kitano, T., Yamamoto, T., Kinoshita, M., 2012. Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases. Development, Growth & Differentiation 54, 546–556. Ansai, S., Sakuma, T., Yamamoto, T., Ariga, H., Uemura, N., Takahashi, R., Kinoshita, M., 2013. Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases. Genetics 193, 739–749. Bibikova, M., Carroll, D., Segal, D.J., Trautman, J.K., Smith, J., Kim, Y.-G., Chandrasegaran, S., 2001. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Molecular and Cellular Biology 21, 289–297. Cade, L., Reyon, D., Hwang, W.Y., Tsai, S.Q., Patel, S., Khayter, C., Joung, J.K., Sander, J.D., Peterson, R.T., Yeh, J.R., 2012. Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic Acids Research 40, 8001–8010. Capecchi, M.R., 2005. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nature Reviews Genetics 6, 507–512. Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N. V., Bogdanove, A.J., Voytas, D.F., 2011. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research 39. Chen, J., Zhang, X., Wang, T., Li, Z., Guan, G., Hong, Y., 2012. Efficient detection, quantification and enrichment of subtle allelic alterations. DNA Research 19, 423–433. Deng, C., Capecchi, M.R., 1992. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Molecular and Cellular Biology 12, 3365–3371. Doyon, Y., McCammon, J.M., Miller, J.C., Faraji, F., Ngo, C., Katibah, G.E., Amora, R., Hocking, T.D., Zhang, L., Rebar, E.J., Gregory, P.D., Urnov, F.D., Amacher, S.L., 2008. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nature Biotechnology 26, 702–708. Guan, G., Yan, Y., Chen, T., Yi, M., Ni, H., Naruse, K., Nagahama, Y., Hong, Y., 2013. Nanos3 gene targeting in medaka ES cells. International Journal of Biological Sciences 9, 444–454. Hong, Y., Winkler, C., Schartl, M., 1998. Production of medakafish chimeras from a stable embryonic stem cell line. Proceedings of the National Academy of Sciences of the United States of America 95, 3679–3684. Hong, N., Li, Z., Hong, Y., 2011. Fish stem cell cultures. International Journal of Biological Sciences 7, 392–402. Jasin, M., 1996. Genetic manipulation of genomes with rare-cutting endonucleases. Trends in Genetics 12, 224–228. Liu, L., Hong, N., Xu, H., Li, M., Yan, Y., Purwanti, Y., Yi, M., Li, Z., Wang, L., Hong, Y., 2009. Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells. Gene Expression Patterns 9, 541–548. Shima, A., Mitani, H., 2004. Medaka as a research organism: past, present and future. Mechanisms of Development 121, 599–604. Thomas, K.R., Capecchi, M.R., 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512. Weidinger, G., Stebler, J., Slanchev, K., Dumstrei, K., Wise, C., Lovell-Badge, R., Thisse, C., Thisse, B., Raz, E., 2003. dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Current Biology 13, 1429–1434. Wittbrodt, J., Shima, A., Schartl, M., 2002. Medaka—a model organism from the Far East. Nature Reviews Genetics 3, 53–64. Xiao, A., Wang, Z., Hu, Y., Wu, Y., Luo, Z., Yang, Z., Zu, Y., Li, W., Huang, P., Tong, X., Zhu, Z., Lin, S., Zhang, B., 2013. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Research 41, e141. Yan, Y., Hong, N., Chen, T., Li, M., Wang, T., Guan, G., Qiao, Y., Chen, S., Schartl, M., Li, C.M., Hong, Y., 2013. p53 gene targeting by homologous recombination in fish ES cells. PLoS One 8, e59400. Zhang, X., Guan, G., Chen, J., Naruse, K., Hong, Y., 2013. Parameters and efficiency of direct gene disruption by zinc finger nucleases in medaka embryos. Marine Biotechnology 16 (NY), 125–134. Zu, Y., Tong, X., Wang, Z., Liu, D., Pan, R., Li, Z., Hu, Y., Luo, Z., Huang, P., Wu, Q., Zhu, Z., Zhang, B., Lin, S., 2013. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nature Methods 10, 329–331.

Direct gene disruption by TALENs in medaka embryos.

Targeted gene disruption (GD) is powerful for generating genetic alterations in animal genomes. Engineered endonucleases such as zinc finger nucleases...
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